PROJECT SUMMARY HIV-1 infection depends on the completion of early steps in the virus replication cycle, including fusion, reverse transcription, uncoating, nuclear entry, and integration. Recent work has indicated that microtubule- dependent intracytoplasmic trafficking, directed by the molecular motors kinesin and dynein, plays an important role in the completion of these processes. We and others have observed that dynein inhibition through small molecule inhibitors, dominant negative proteins, and siRNA treatment, results in decreased HIV infection. Dynein has also been reported to influence HIV-1 uncoating and reverse transcription. An understanding of the specific interactions between HIV-1 and the dynein motor complex should facilitate therapeutic targeting of early post-entry steps in HIV-1 infection. Key unanswered questions regarding the mechanism of dynein- dependent trafficking are: (1) How do HIV-1 particles engage the transport machinery? (2) What viral proteins are involved? (3) What mechanism is required for the cellular proteins to traffic HIV? My project will begin to answer these questions using a combination of biochemical, genetic, and microscopy approaches. Dynein adaptor proteins activate dynein-dependent transport, and link cargo to dynein to provide specificity. Through an siRNA screen, I have determined that the dynein adaptor BICD2 and dynactin components are necessary for HIV-1 infection. I have also observed that the HIV-1 capsid is able to bind the dynein complex in vitro, suggesting that this interaction may play a role in trafficking. We have also seen that binding of cyclophilin A to the HIV-1 capsid reduces the potency of inhibition of HIV-1 infection by the dynein inhibitor ciliobrevin D. This observation suggests that the capsid-cyclophilin A interaction is important for efficient HIV-1 utilization of dynein. These findings lead to the hypothesis that BICD2, dynactin and cyclophilin A are acting together to form a complex between the HIV capsid and dynein for trafficking and infection. To test this, I propose two specific aims. In Specific Aim 1, I will determine the role of BICD2, dynactin, and cyclophilin A in HIV trafficking and infection by assessing the necessity for these proteins in trafficking, and HIV capsid binding. I will also determine the stage of infection (fusion, reverse transcription, nuclear entry, integration) affected by depletion of these proteins. In Specific Aim 2, I will determine the necessary interactions of the dynein complex and the HIV-1 capsid. To do this, binding of the HIV capsid to BICD2, dynactin, and cyclophilin A will be assessed. Additionally, I will determine if these components are necessary for the HIV capsid-dynein binding seen previously. Finally, I will determine the necessary regions of these proteins for the binding, and determine if this binding is necessary for trafficking and infection. The work will define the direct interactions between HIV and the dynein complex and the role of these interactions in dynein localization, HIV-1 trafficking, and infection.